Project Summary/Abstract Patients suffering from facial neuropathic pain commonly experience sensations of pain in the absence of any relevant environmental stimulus. The identity of the neurons responsible for generating this spontaneous pain remains unresolved, creating a barrier to rational targeting of treatment. The scientific goal of this proposal is to establish the neuronal substrates responsible for initiating stimulus-independent neuropathic pain. In previous post-doctoral work, the candidate has developed an in vivo calcium imaging technique to monitor activity in large populations of genetically defined trigeminal neurons in mice, and in AIM 1 will apply this approach to define the transcriptional identity and injury status of primary sensory neurons giving rise to spontaneous activity in a model of trigeminal nerve injury. Genetic access to the injury status of sensory neurons has been achieved through generation of a novel mouse line expressing CreERT2 from the Atf3 locus, directing expression specifically to injured neurons. AIM 2 will explicitly investigate the causal link between ectopic activity in specific sensory populations and manifestations of spontaneous pain. Output from defined sensory populations will be silenced through genetically directed inhibition of synaptic transmission, and the resulting effect on behavioral and physiological surrogates of stimulus-independent pain sensation will be assessed. These experiments will rely on extensive training in pain-related behavioral paradigms by the primary mentor, with assistance from the BCH Pain Core. The results of AIMs 1 & 2 will identify peripheral generators of spontaneous neuropathic pain. AIM 3 will examine nerve injury-induced spontaneous activity in the medullary dorsal horn, the first central area processing craniofacial pain information, and dissect the functional relationship between central spontaneous activity and specific peripheral inputs. This will rely on novel techniques to perform calcium imaging in the brainstem of awake behaving animals, which will be developed in close consultation with Dr. Mark Andermann. The mentored phase of this award will be supervised by an advisory committee of Drs. Clifford Woolf, Mark Andermann, David Ginty and Stephen Liberles. An integrated training plan provides intensive training in the modeling and measurement of neuropathic pain, design and analysis of assays for pain behavior, scientific leadership skills, and responsible conduct of research, thus providing a platform for successful transition to direction of an independent research program investigating mechanisms of normal and pathological pain.